title: Nitric oxide synthase dysfunction contributes to impaired cerebroarteriolar reactivity in experimental cerebral malaria.
abstract: Cerebrovascular dysfunction plays a key role in the pathogenesis of cerebral malaria. In experimental cerebral malaria (ECM) induced by Plasmodium berghei ANKA, cerebrovascular dysfunction characterized by vascular constriction, occlusion and damage results in impaired perfusion and reduced cerebral blood flow and oxygenation, and has been linked to low nitric oxide (NO) bioavailability. Here, we directly assessed cerebrovascular function in ECM using a novel cranial window method for intravital microscopy of the pial microcirculation and probed the role of NOS isoforms and phosphorylation patterns in the impaired vascular responses. We show that pial arteriolar responses to endothelial NOS (eNOS) and neuronal NOS (nNOS) agonists (Acetylcholine (ACh) and N-Methyl-D-Aspartate (NMDA)) were blunted in mice with ECM, and could be partially recovered by exogenous supplementation of tetrahydrobiopterin (BH4). Pial arterioles in non-ECM mice infected by Plasmodium berghei NK65 remained relatively responsive to the agonists and were not significantly affected by BH4 treatment. These findings, together with the observed blunting of NO production upon stimulation by the agonists, decrease in total NOS activity, augmentation of lipid peroxidation levels, upregulation of eNOS protein expression, and increase in eNOS and nNOS monomerization in the brain during ECM development strongly indicate a state of eNOS/nNOS uncoupling likely mediated by oxidative stress. Furthermore, the downregulation of Serine 1176 (S1176) phosphorylation of eNOS, which correlated with a decrease in cerebrovascular wall shear stress, implicates hemorheological disturbances in eNOS dysfunction in ECM. Finally, pial arterioles responded to superfusion with the NO donor, S-Nitroso-L-glutathione (GSNO), but with decreased intensity, indicating that not only NO production but also signaling is perturbed during ECM. Therefore, the pathological impairment of eNOS and nNOS functions contribute importantly to cerebrovascular dysfunction in ECM and the recovery of intrinsic functionality of NOS to increase NO bioavailability and restore vascular health represents a target for ECM treatment.
authors.affiliation: Center for Malaria Research, La Jolla Bioengineering Institute, San Diego, California, United States of America. pkong@ljbi.org
authors.forename: Peng Kai
authors.initials: PK
authors.lastname: Ong
authors.forename: Benoît
authors.initials: B
authors.lastname: Melchior
authors.forename: Yuri C
authors.initials: YC
authors.lastname: Martins
authors.forename: Anthony
authors.initials: A
authors.lastname: Hofer
authors.forename: Pamela
authors.initials: P
authors.lastname: Orjuela-Sánchez
authors.forename: Pedro
authors.initials: P
authors.lastname: Cabrales
authors.forename: Graziela M
authors.initials: GM
authors.lastname: Zanini
authors.forename: John A
authors.initials: JA
authors.lastname: Frangos
authors.forename: Leonardo J M
authors.initials: LJ
authors.lastname: Carvalho
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives / Q000494:pharmacology; D002560:Cerebrovascular Circulation; D018679:Cholinergic Agonists; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D005260:Female; D016779:Malaria, Cerebral / Q000378:metabolism / Q000469:parasitology / Q000503:physiopathology; D051379:Mice; D008833:Microcirculation; D016202:N-Methylaspartate / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D010962:Plasmodium berghei / Q000378:metabolism*
chemical_list: D018679:Cholinergic Agonists; D018690:Excitatory Amino Acid Agonists; D001708:Biopterins; D009569:Nitric Oxide; D016202:N-Methylaspartate; D052248:Nitric Oxide Synthase Type I; D052250:Nitric Oxide Synthase Type III; C496321:Nos1 protein, mouse; C496315:Nos3 protein, mouse; C003402:sapropterin; D000109:Acetylcholine